CC LAB EXPE 8 Flashcards
Amylase was first measured quantitatively by an _____ introduced by ______
Iodometric method; by Wohlegemuth
Who introduced a procedure in 1938 that standardized the amounts of starch and iodine?
Somogyi
His work became the basis for the widely used amyloclastic and saccharogenic methods introduced in 1956 and 1960
Somogyi
What are the disadvantages of the methods used by wohlegemuth and Somogyi?
Long incubation times, endogenous glucose interference, and unstable reaction colors resulting in poor reproducibility and reliability
Wallenfels et Al introduced____ as defined substrates for amylase determination in a procedure that eliminated interference from endogenous glucose and pyruvate
P-nitrophenylglycosides
The present procedure is based on modifications of Wallenfels, using a substrate ________ with the terminal glucose blocked to reduce spontaneous degradation of the substrate by ____ and _____
P-nitrophenyl- D-maltoheptaoside (PNPG7); glucosidase and glucoamylase
The determination of amylase activity in serum and and urine is most commonly performed for the diagnosis of _____
Acute pancreatitis
True or False: in acute pancreatitis, amylase levels are elevated for longer periods of time in serum than in urine
False: in acute pancreatitis, amylase levels are elevated for longer periods of time in urine than in serum
_____ hydrolyzes PNPG7 to PNPG3 and _____
Amylase; Maltotetraose
____ hydroxides PNPG3 to PNPG1 and glucose
Glucoamylase
___ hydrolyzed to glucosidase to glucose and p-nitrophenol, which produces a _____ color
PNPG1; Yellow color
The rate of increase in absorbance of p-nitrophenol is measured at _____ and is ____ to the amylase activity in the sample
405nm; directly proportional
PNPG3 +____
Matlotetraose
PNPG1 +____
Glucose
P-nitrophenol + ____
Glucose
Assay for amylase involves the determination of both ______ and the _____
Salivary amylase and pancreatic amylase
This method measure the breakdown of starch substrate
Amyloclastic method
What are under amyloclastic methods
Viscosimetric
Iodometric
Turbidimetric
Nephelometric
An instrument used to measure the change in flow rate of starch
Viscosimeter
This procedure involves the hydrolysis of starch that results in the disruption of its molecular structure with reduction of viscosity
Viscosimetric
This method are very inaccurate, highly dependent on the nature of starch substrate and unsuitable for the measurement of low amylase activities
Viscosimetric
This procedure measure the decrease in absorbance of a turbid starch substrate due to reduction in starch granule size as a result of amylase activity.
Turbidimetric
These methods are simple and rapid, but poor in precision at near normal activity of amylase and inadequate substrate stability.
Turbidimetric
True or False: the Turbidimetric change is linearly related to amylase activity, except perhaps in the first two minutes of the reaction
The Turbidimetric change is NOT linearly related to amylase activity, except perhaps in the first two minutes of the reaction
What procedure involves; The endpoint is indicated by the absence of the blue-starch iodine color
Iodometric
Only___ gives a blue color with iodine, ____ forms a weak red color
Amylose; Amylopectin
The characteristic blue color is due to the helical configuration which amylose forms with iodine; each turn of helix contains ___ glucose units and surrounds __ molecule of iodine
6 glucose units and 1molecule of iodine
The blue color is obtained only when the overall chain length of amylose is greater than ____
45 glucose units
What are the disadvantages of Iodometric Methods
-only the hydrolysis of the amylose portion of the starch is being measured yet amylase has greater activity in hydrolyzing amylopectin
- starches of different origin vary in their proportions of amylose and amylopectin
- amylase does not act under the substrate saturation conditions
- interference from proteins, particularly albumin, and from lipids
___ based on reduction of light-scattering of starch substrate by amylase activity
Nephelometric assays
What are the advantages of Nephelometric Approach
Simplicity, Speed, Precision, and Zero Order kinetics
It is claimed to be suitable for emergency purposes, results being available in less than 3 minutes with direct read-out, bench working time and manipulation are minimal, sample blanking is NOT required
Nephelometric
In this method, a starch bound to a dye is hydrolyzed by amylase with the release of dye-substrate fragments which are water soluble
Chromogenic Methods
This method is based on the hydrolysis of starch by amylase to produce carbohydrates that have reducing properties
Saccharogenic Method
To convert Somogyi units to IU, multiply values by _____
1.85
This involves the measurement of amylase activity by HLPC with fluorescence detection, using a fluorogenic substrate
Fluorogenic methods
HPLC product analysis revealed a treated production of substituted oligosaccharide by ____ than by ____, and this observation was used as a the basis of a differential assay for these isoamylases in ____
Pancreatic; salivary amylase ; Human serum
This method involves the usage of fluorescein-labeled amylose
Fluorescence Depolarization Method
Fluorescence Depolarization Method: the degree of polarization of light emitted from this fluorophore when excited by a polarized beam of light is ____ by amylase ____
Decreased; hydrolysis
True or False: (Fluorescence Depolarization Method) : The smaller molecules formed are less polarized because they rotate faster than the intact substrate
True
This method is the basis of the Abbott TDX Amylase assay and this method provides a simple and sensitive direct assay but requires special instrumentation
Fluorescence Depolarization Method
This method is for measuring amylase activity using radio labeled starch substrate
Radiometric Method
Amylase Hydrolysis of the radio-labeled substrate releases ____ which is ___ proportional to amylase activity
Free radioisotope; proportional
This method involves immunoassay using polyclonal antibodies produced against salivary or pancreatic isoamylase as antigen
Immunologic Method
Maltopentose ——> maltotriose + maltose
Amylase
Maltotriose + maltose ——> glucose
Alpha glucosidase
Glucose + ATP ——-> glucose-6-phosphate + ADP
Hexokinase
Glucose-6-phosphate + NAD ——-> 6-phosphogluconolactone + NADH
G6PD
The IFCC has optimized this method at 37C, recommending it as a reference method for amylase determination
5 ethylidene- 4- nitrophenyl glycosides as subtrates
This involves the use of 2-chloro-p-nitrophenol as an indicator
Use of 2-chloro-p-nitrophenyl- alpha-D-maltotrioside as substrate
Disadvantages of 2-chloro-p-nitrophenyl-D-alpha-matlotrioside as a subtracted
Slow reaction rate
Variation in molar absorptivity of chloro-p-nitrophenol associated with changes in pH
Temperature
Protein content
Presence of potassium thiocyanate = allosteric changes to amylase and precluding the use of antibodies for p-amylase determination
True or false: In amylase determination, heparinized plasma can also be used
True
Anticoagulants such as ______, ______, and _____ should NOT be used as these bind to calcium (an ion needed for amylase activity) , this May lower amylase activity (EDTA by 10%)
Citrate
EDTA
Oxalate
Urine specimens should be collected for a ____, adjusted to a pH of ___ with either 0.1N NaOH or 0.1 HCl and kept refrigerated until assayed
24 hour period, ph 7
Amylase in serum and urine is reported stable for ____ at ____ and for several months when stored at _____ degrees Celsius and protected against evaporation and bacterial contamination.
1 week at RT; 2-8C
Stability may decrease in urine samples with ph less than ____
5
What causes the increase in pancreatic amylase in serum?
Macroamylasemia
Lipemia
Bilirubin concentration greater than 20 mg/dL (falsely increase)
Insulin and some bacteria
Administration of morphine and opiates (falsely increase)
Contamination of sample with ___ and ____ should be avoided as these contain alpha-amylase
Sweat and saliva
What is the linearity of the method?
1,500 IU/L
What are the isoenzymes of amylase?
Pancreatic and Salivary fractions
What methods can be used to differentiate the isoenzymes of amylase?
Electrophoresis
Ion exchange chromatography
Isoelectric focusing
Selective inhibition of S-AMY by a wheat germ inhibitor
Immuno precipitation by a monoclononal antibody
Immune inhibition
Only methods based on selective isoenzyme inhibition by monoloclonal antibodies have shown sufficient——-
Precision
Reliability
Practicability
Analytical speed to allow the introduction of P-AMY determination into a clinical practice
WHAT IS ACCR?
Amylase-creatinine clearance ratio
It was established as an index for screening of acute pancreatitis
ACCR (Amylase-Creatinine Clearance Ratio)
ACCR is increased in cases of _______________
Diabetes keto acidosis
Renal insufficiency
Severe burns
Reference range of ACCR?
1-5%
Reagent composition of AMYLASE DETERMINATION
P-nitrophenyl-D-Maltoheptaoside
Glucosidase-yeast
Glucoamylase- rhizopus spp.
Sodium chloride
Calcium chloride
Buffer
PH
P-nitrophenyl- D- Maltoheptaoside (____)
0.9 mM
Glucosidase- yeast (_____)
25, 000 IU/L
Glucoamylase- rhizopus spp (___)
10,000 IU/L
Sodium chloride [____]
50mM
Calcium chloride (_____)
5 mM
Buffer (___)
50 mM
Ph [______]
6.9
TV=
1.025
MMA=
8.5
SV=
0.025 mL
Multiplication factor of AMYLASE
4824
Linearity of amylase
1,500 IU/L
Expected values in serum
Up to 96 IU/L
Expected values in urine
18-330 IU/L
